TY - JOUR A1 - Rauschenberger, Vera A1 - Piro, Inken A1 - Kasaragod, Vikram Babu A1 - Hörlin, Verena A1 - Eckes, Anna-Lena A1 - Kluck, Christoph J. A1 - Schindelin, Hermann A1 - Meinck, Hans-Michael A1 - Wickel, Jonathan A1 - Geis, Christian A1 - Tüzün, Erdem A1 - Doppler, Kathrin A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation JF - Frontiers in Molecular Neuroscience N2 - Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor’s glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera. KW - glycine receptor KW - autoantibodies KW - glycosylation KW - extracellular domain KW - adsorption Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304206 VL - 16 ER - TY - JOUR A1 - Hecker, Katharina A1 - Grüner, Julia A1 - Hartmannsberger, Beate A1 - Appeltshauser, Luise A1 - Villmann, Carmen A1 - Sommer, Claudia A1 - Doppler, Kathrin T1 - Different binding and pathogenic effect of neurofascin and contactin–1 autoantibodies in autoimmune nodopathies JF - Frontiers in Immunology N2 - Introduction IgG4 autoantibodies against paranodal proteins are known to induce acute-onset and often severe sensorimotor autoimmune neuropathies. How autoantibodies reach their antigens at the paranode in spite of the myelin barrier is still unclear. Methods We performed in vitro incubation experiments with patient sera on unfixed and unpermeabilized nerve fibers and in vivo intraneural and intrathecal passive transfer of patient IgG to rats, to explore the access of IgG autoantibodies directed against neurofascin-155 and contactin-1 to the paranodes and their pathogenic effect. Results We found that in vitro incubation resulted in weak paranodal binding of anti-contactin-1 autoantibodies whereas anti-neurofascin-155 autoantibodies bound to the nodes more than to the paranodes. After short-term intraneural injection, no nodal or paranodal binding was detectable when using anti-neurofascin-155 antibodies. After repeated intrathecal injections, nodal more than paranodal binding could be detected in animals treated with anti-neurofascin-155, accompanied by sensorimotor neuropathy. In contrast, no paranodal binding was visible in rats intrathecally injected with anti-contactin-1 antibodies, and animals remained unaffected. Conclusion These data support the notion of different pathogenic mechanisms of anti-neurofascin-155 and anti-contactin-1 autoantibodies and different accessibility of paranodal and nodal structures. KW - autoimmune nodopathy KW - IgG4 KW - neurofascin KW - contactin KW - node of ranvier KW - inflammatory neuropathy KW - passive transfer Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320395 VL - 14 ER - TY - INPR A1 - Brenner, Marian A1 - Zink, Christoph A1 - Witzinger, Linda A1 - Keller, Angelika A1 - Hadamek, Kerstin A1 - Bothe, Sebastian A1 - Neuenschwander, Martin A1 - Villmann, Carmen A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - 7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase T2 - eLife N2 - Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain. KW - 7,8-dihydroxyflavone (7,8-DHF) KW - pyridoxal phosphatase (PDXP) KW - vitamin B6 KW - PDXP inhibitors Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350446 ER - TY - JOUR A1 - Wiessler, Anna-Lena A1 - Talucci, Ivan A1 - Piro, Inken A1 - Seefried, Sabine A1 - Hörlin, Verena A1 - Baykan, Betül B. A1 - Tüzün, Erdem A1 - Schaefer, Natascha A1 - Maric, Hans M. A1 - Sommer, Claudia A1 - Villmann, Carmen T1 - Glycine receptor β–targeting autoantibodies contribute to the pathology of autoimmune diseases JF - Neurology: Neuroimmunology & Neuroinflammation N2 - Background and Objectives Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα subunit–binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyRβ subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology. Methods In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRβ. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRβ binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyRβ aAb binding were resolved by whole-cell patch-clamp recordings. Results Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRβ aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRβ colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRβ aAb from both patients to its target impair glycine efficacy. Discussion Our study establishes GlyRβ as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRβ impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRβ aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization. KW - autoantibody (aAb) KW - glycine receptor (GlyR) KW - stiff-person syndrome (SPS) KW - clinical neurology KW - movement disorders KW - progressive encephalitis with rigidity and myoclonus (PERM) Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349958 VL - 11 IS - 2 ER - TY - JOUR A1 - Drehmann, Paul A1 - Milanos, Sinem A1 - Schaefer, Natascha A1 - Kasaragod, Vikram Babu A1 - Herterich, Sarah A1 - Holzbach-Eberle, Ulrike A1 - Harvey, Robert J. A1 - Villmann, Carmen T1 - Dual role of dysfunctional Asc-1 transporter in distinct human pathologies, human startle disease, and developmental delay JF - eNeuro N2 - Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5. Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5′ and 3′ untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1\(^{G307R}\) from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1\(^{G307R}\) did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay. KW - Asc-1 transporter KW - candidate gene KW - glycine receptor KW - glycine uptake KW - human startle disease KW - NMDAR Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349947 VL - 10 IS - 11 ER - TY - JOUR A1 - Janzen, Dieter A1 - Bakirci, Ezgi A1 - Faber, Jessica A1 - Andrade Mier, Mateo A1 - Hauptstein, Julia A1 - Pal, Arindam A1 - Forster, Leonard A1 - Hazur, Jonas A1 - Boccaccini, Aldo R. A1 - Detsch, Rainer A1 - Teßmar, Jörg A1 - Budday, Silvia A1 - Blunk, Torsten A1 - Dalton, Paul D. A1 - Villmann, Carmen T1 - Reinforced Hyaluronic Acid-Based Matrices Promote 3D Neuronal Network Formation JF - Advanced Healthcare Materials N2 - 3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30–500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca\(^{2+}\) imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases. KW - 3D model systems KW - melt electrowriting KW - cortical neurons KW - astrocytes KW - Ca\(^{2+}\)-Imaging KW - hyaluronic acid Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318682 VL - 11 IS - 21 ER - TY - JOUR A1 - Schaefer, Natascha A1 - Roemer, Vera A1 - Janzen, Dieter A1 - Villmann, Carmen T1 - Impaired Glycine Receptor Trafficking in Neurological Diseases JF - Frontiers in Molecular Neuroscience N2 - Ionotropic glycine receptors (GlyRs) enable fast synaptic neurotransmission in the adult spinal cord and brainstem. The inhibitory GlyR is a transmembrane glycinegated chloride channel. The immature GlyR protein undergoes various processing steps, e.g., folding, assembly, and maturation while traveling from the endoplasmic reticulum to and through the Golgi apparatus, where post-translational modifications, e.g., glycosylation occur. The mature receptors are forward transported via microtubules to the cellular surface and inserted into neuronal membranes followed by synaptic clustering. The normal life cycle of a receptor protein includes further processes like internalization, recycling, and degradation. Defects in GlyR life cycle, e.g., impaired protein maturation and degradation have been demonstrated to underlie pathological mechanisms of various neurological diseases. The neurological disorder startle disease is caused by glycinergic dysfunction mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). In vitro studies have shown that most recessive forms of startle disease are associated with impaired receptor biogenesis. Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies. Binding of GlyR autoantibodies leads to enhanced receptor internalization. Here we focus on the normal life cycle of GlyRs concentrating on assembly and maturation, receptor trafficking, post-synaptic integration and clustering, and GlyR internalization/recycling/degradation. Furthermore, this review highlights findings on impairment of these processes under disease conditions such as disturbed neuronal ER-Golgi trafficking as the major pathomechanism for recessive forms of human startle disease. In SPS, enhanced receptor internalization upon autoantibody binding to the GlyR has been shown to underlie the human pathology. In addition, we discuss how the existing mouse models of startle disease increased our current knowledge of GlyR trafficking routes and function. This review further illuminates receptor trafficking of GlyR variants originally identified in startle disease patients and explains changes in the life cycle of GlyRs in patients with SPS with respect to structural and functional consequences at the receptor level. KW - glycine receptor KW - startle disease KW - autoimmune antibodies KW - protein maturation KW - trafficking pathways Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227531 VL - 11 IS - 291 ER - TY - JOUR A1 - Janzen, Dieter A1 - Slavik, Benedikt A1 - Zehe, Markus A1 - Sotriffer, Christoph A1 - Loos, Helene M. A1 - Buettner, Andrea A1 - Villmann, Carmen T1 - Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABA\(_{A}\) receptor function in vitro JF - Journal of Neurochemistry N2 - Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABA\(_{A}\) receptors (GABA\(_{A}\)Rs). In this study, the modulatory potential of 11 SQTs at GABA\(_{A}\)Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole-cell recordings. Significantly different effects of β-caryophyllene and α-humulene, as well as their respective derivatives β-caryolanol and humulol, were observed in the HEK293 cell system. In neurons, the concomitant presence of phasic and tonic GABA\(_{A}\)R configurations accounts for differences in receptor modulation by SQTs. The in vivo presence of the γ\(_{2}\) and δ subunits is important for SQT modulation. While phasic GABA\(_{A}\) receptors in hippocampal neurons exhibited significantly altered GABA-evoked current amplitudes in the presence of humulol and guaiol, negative allosteric potential at recombinantly expressed α\(_{1}\)β\(_{2}\)γ\(_{2}\) receptors was only verified for humolol. Modeling and docking studies provided support for the binding of SQTs to the neurosteroid-binding site of the GABA\(_{A}\)R localized between transmembrane segments 1 and 3 at the (\(^{+}\)α)-(\(^{-}\)α) interface. In sum, differences in the modulation of GABA\(_{A}\)R isoforms between SQTs were identified. Another finding is that our results provide an indication that nutritional digestion affects the neurotropic potential of natural compounds. KW - allosteric modulation KW - GABA\(_{A}\) receptor KW - patch clamp recording Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259546 VL - 159 IS - 1 ER - TY - JOUR A1 - Fischhaber, Natalie A1 - Faber, Jessica A1 - Bakirci, Ezgi A1 - Dalton, Paul D. A1 - Budday, Silvia A1 - Villmann, Carmen A1 - Schaefer, Natascha T1 - Spinal Cord Neuronal Network Formation in a 3D Printed Reinforced Matrix-A Model System to Study Disease Mechanisms JF - Advanced Healthcare Materials N2 - 3D cell cultures allow a better mimicry of the biological and mechanical environment of cells in vivo compared to 2D cultures. However, 3D cell cultures have been challenging for ultrasoft tissues such as the brain. The present study uses a microfiber reinforcement approach combining mouse primary spinal cord neurons in Matrigel with melt electrowritten (MEW) frames. Within these 3D constructs, neuronal network development is followed for 21 days in vitro. To evaluate neuronal development in 3D constructs, the maturation of inhibitory glycinergic synapses is analyzed using protein expression, the complex mechanical properties by assessing nonlinearity, conditioning, and stress relaxation, and calcium imaging as readouts. Following adaptation to the 3D matrix-frame, mature inhibitory synapse formation is faster than in 2D demonstrated by a steep increase in glycine receptor expression between days 3 and 10. The 3D expression pattern of marker proteins at the inhibitory synapse and the mechanical properties resemble the situation in native spinal cord tissue. Moreover, 3D spinal cord neuronal networks exhibit intensive neuronal activity after 14 days in culture. The spinal cord cell culture model using ultrasoft matrix reinforced by MEW fibers provides a promising tool to study and understand biomechanical mechanisms in health and disease. KW - 3D cell cultures KW - spinal cord neurons KW - neuronal networks KW - mouse Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256353 VL - 10 IS - 19 ER - TY - JOUR A1 - Piro, Inken A1 - Eckes, Anna-Lena A1 - Kasaragod, Vikram Babu A1 - Sommer, Claudia A1 - Harvey, Robert J. A1 - Schaefer, Natascha A1 - Villmann, Carmen T1 - Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease JF - Frontiers in Molecular Neuroscience N2 - Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC\(_{50}\) value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations. KW - glycine receptor KW - hyperekplexia KW - startle disease KW - gephyrin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246676 SN - 1662-5099 VL - 14 ER -